Functional Green-Tuned Proteorhodopsin from Modern Stromatolites
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RESEARCH ARTICLE Functional Green-Tuned Proteorhodopsin from Modern Stromatolites Virginia Helena Albarracín1,2*, Ivana Kraiselburd3, Christian Bamann4, Phillip G. Wood4, Ernst Bamberg4, María Eugenia Farias4, Wolfgang Gärtner5* 1 Planta Piloto de Procesos Industriales y Microbiológicos (PROIMI), CCT, CONICET. Av. Belgrano y Pasaje Caseros. 4000- S. M. de Tucumán, Argentina, 2 Facultad de Ciencias Naturales e Instituto Miguel Lillo, Universidad Nacional de Tucumán, 4000, S. M. de Tucumán, Argentina, 3 Instituto de Biología Molecular y Celular de Rosario (IBR - CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (FBIOYF - UNR), Suipacha 590, 2000, Rosario, Santa Fe, Argentina, 4 Max-Planck-Institute for Biophysics, Max-von-Laue-Straße 3, D-60438 Frankfurt am Main, Germany, 5 Max-Planck-Institute for Chemical Energy a11111 Conversion, Stiftstrasse 34–36, D-45470 Mülheim, Germany * [email protected] (VHA); [email protected] (WG) Abstract OPEN ACCESS The sequenced genome of the poly-extremophile Exiguobacterium sp. S17, isolated from modern stromatolites at Laguna Socompa (3,570 m), a High-Altitude Andean Lake (HAAL) Citation: Albarracín VH, Kraiselburd I, Bamann C, Wood PG, Bamberg E, Farias ME, et al. (2016) in Argentinean Puna revealed a putative proteorhodopsin-encoding gene. The HAAL area Functional Green-Tuned Proteorhodopsin from is exposed to the highest UV irradiation on Earth, making the microbial community living in Modern Stromatolites. PLoS ONE 11(5): e0154962. the stromatolites test cases for survival strategies under extreme conditions. The heterolo- doi:10.1371/journal.pone.0154962 gous expressed protein E17R from Exiguobacterium (248 amino acids, 85% sequence Editor: Erika Kothe, Friedrich Schiller University, identity to its ortholog ESR from E. sibiricum) was assembled with retinal displaying an GERMANY absorbance maximum at 524 nm, which makes it a member of the green-absorbing PR-sub- Received: February 9, 2016 family. Titration down to low pH values (eventually causing partial protein denaturation) indi- Accepted: April 21, 2016 cated a pK value between two and three. Global fitting of data from laser flash-induced Published: May 17, 2016 absorption changes gave evidence for an early red-shifted intermediate (its formation being below the experimental resolution) that decayed (τ1 =3.5μs) into another red-shifted inter- Copyright: © 2016 Albarracín et al. This is an open τ μ τ access article distributed under the terms of the mediate. This species decayed in a two-step process ( 2 =84 s, 3 = 11 ms), to which the Creative Commons Attribution License, which permits initial state of E17-PR was reformed with a kinetics of 2 ms. Proton transport capability of unrestricted use, distribution, and reproduction in any the HAAL protein was determined by BLM measurements. Additional blue light irradiation medium, provided the original author and source are reduced the proton current, clearly identifying a blue light absorbing, M-like intermediate. credited. The apparent absence of this intermediate is explained by closely matching formation and Data Availability Statement: All relevant data are decay kinetics. within the paper and its Supporting Information files. Funding: This work was supported by the PICT 2010 2991 Project (FONCyT, Argentina), PIP-CONICET 2013-0519 Project, the Proalar Agreement (MINCYT- DAAD) DA/13/01, The Max-Planck-Society (MPI for Biophysics and MPI for Chemical Energy Conversion, Introduction Germany). A Marie Curie FP7-People-2010-IIF Microbial rhodopsins are considered to be the simplest energy-harvesting photoreceptors con- EXTREMOPHIL (273831) in Germany and its return phase in Argentina (PIIFR-GA-2010-910831- sisting of a single, membrane-embedded protein covalently binding a retinal (Vitamin-A alde- EXTREMOPHIL) supported research work of VHA. hyde) chromophore [1]. The study of the microbial archetype, bacteriorhodopsin (BR) from The funders had no role in study design, data the archaeon Halobacterium salinarum [2], led to the suggestion that BR evolved as a PLOS ONE | DOI:10.1371/journal.pone.0154962 May 17, 2016 1/18 proteorhodopsin from Modern Stromatolites collection and analysis, decision to publish, or consequence of exposure to the extreme halophilic, oxygen-deprived environment of H. sali- preparation of the manuscript. narum. However, further surveys demonstrated the worldwide existence of many microbial Competing Interests: The authors have declared rhodopsins with a spectrum of functions in ecological niches, thus being probably the most that no competing interests exist. abundant phototrophic system on Earth [3]. Out of this versatile family of proteins, the proteorhodopsins (PRs), bacterial ion pumps, represent the largest group [3]. They were mostly found in microbial communities living in environments where solar exposure is high i.e. the upper layer of oceans, freshwater, brackish and hypersaline aquatic ecosystems [4–18], sea-ice matrix [19], non-marine permafrost [20, 21] and phyllosphere [22]. Moreover, proteorhodopsin genes are widely distributed among divergent bacterial taxa included alpha, beta, delta and gamma proteobacteria, Bacteroidetes, Flavobacteria, Firmicutes, Deinococcus-Thermus, Chloroflexi, Planctomycetes and Actinobac- teria [23–28]. Detailed molecular studies on various PRs and their three-dimensional structure [21, 29] (in addition to the enormous information on structure and function of BR) have generated a precise picture of their light-induced reactions [3]. Key amino acids, already identified as instrumental for the function of BR have been allocated also to the PRs [30–32]. A further evo- lutionary aspect of PRs was detected from sequence alignments, together with spectroscopic measurements. Two subfamilies of PRs were identified, distinguished by their absorbance max- ima [33–35]. The group coined GPRs (green-absorbing) showed absorbance maxima around 525 nm, whereas the other group near 490 nm (BPRs, blue-absorbing). This switch in absor- bance maxima is accomplished by a single amino acid exchange and is understood as an adap- tation to the prevailing spectral conditions at different depths in a water column with green light dominating close to the surface and solely blue light remaining at greater water depths [33–35]. Microbes might profit from PRs in a variety of ways; planktonic ones may gain a competi- tive advantage by using these photoproteins to generate a light-driven proton gradient for the synthesis of ATP [36, 37]. In extreme environments though, PRs may even be paramount to counteract starvation and other hostile conditions thanks to the potential for harvesting extra energy by means of phototrophy [16, 19]. A characteristic example of a highly irradiated, extreme environment is the High-Altitude Andean Lakes (HAAL) area in the Central Andes region in South America [38, 39]. These shal- low lakes and salterns dispersed at altitudes above 3,000 m are exposed to a unique combina- tion of severe conditions including high solar global and UV irradiation, hypersalinity, wide fluctuations in daily temperatures, desiccation, high pH, and high concentrations of toxic ele- ments including arsenic, which have brought forth a distinguished microbiodiversity of extremophiles [38–42,62,63]. Among microbialites of the HAAL [41, 43–47], modern stromatolites were first described as thriving in the shore of Laguna Socompa, a hypersaline and moderate alkaline lake located at 3,570 m at the base of the active Socompa volcano. These stromatolites (41) exhibit an atypi- cal microbial community with abundant representatives of Deinococcus-Thermus, Rhodobac- teraceae, Desulfobacterales and Spirochaetes. Their metagenome revealed a high proportion of sequences that could not be classified at phylum level, depicting less than 80% identity to the best hit in the NCBI database, suggesting the presence of novel distant lineages [41]. Thus, the study of microbes associated with Socompa stromatolites and their environment could provide further understanding of their adaptation and the function of proteins working under extremely harsh conditions. Several bacterial strains from Socompa stromatolites were isolated, physiologically charac- terized and subjected to genome pyrosequencing [18, 48, 49]. Screening of the genomes of these strains i.e. Exiguobacterium sp. S17, Sphingomonas sp. S17 and Salinivibrio spp. S10B and PLOS ONE | DOI:10.1371/journal.pone.0154962 May 17, 2016 2/18 proteorhodopsin from Modern Stromatolites S34, yielded genes putatively encoding essential traits for survival under multiple environmen- tal extreme conditions, e.g., high levels of UV radiation, elevated salinity, and the presence of poisoning arsenic concentrations [18, 45, 48–50]. Likewise, genes putatively coding for the bacteriorhodopsin protein family were found showing sequential features indicating the pres- ence of proteorhodopsins and xantorhodopsins [18, 45–47]. The former findings called for a more detailed, molecular investigation of the mechanisms involved in the resistance of these strains to extreme but common impairing factors in its original environment. We herein pres- ent the first functional characterization of E17R, a PR from Exiguobacterium sp. S17, a haloto- lerant, highly arsenic resistant extremophile isolated from a modern stromatolite, located at an altitude of more than 3,500 m above sea level. Materials and Methods Strain, media and culture conditions The extremophile Exiguobacterium sp. S17 was previously isolated from modern